The cellular events and molecular pathways leading to the detection, checkpoint response, and repair of spontaneous and double strand break induced DNA damage are not completely known. Although great strides have been made over the last few years, important repair pathways and genetic interactions are still being uncovered. A full understanding of all the genetic interactions in this area is an important goal, since DNA damage repair pathways are often genetically altered in cancer cells. Indeed, many of the genes involved in these pathways are themselves targets of chemotherapeutic drugs. The proposed work will reveal novel genetic relationships and pathways important for DNA replication, recombination, and repair, through a series of genetic and cell biological experiments. Some experiments will explore the pathways involved in double strand break repair by performing a comprehensive epistasis analysis of DNA damage sensitive mutants. The epistasis relationship amongst these genes will reveal which pathways they affect. Gene copy-number and expression data, from over 9,000 tumors profiled by The Cancer Genome Atlas (TCGA), and METABRIC consortiums, were used to prioritize overexpression studies of 93 genes involved in DNA replication, recombination and repair (3R) that are commonly overexpressed in cancer. Overexpression of gene products often result in different genetic imbalances compared to loss of function mutants. The proposed work will model the consequences of overexpressing 3R genes by constructing an overexpression based genetic interactome for cancer-relevant 3R genes, and thereby uncover novel pathways involved in replication, recombination, and repair. Important processes in DNA repair are homologous recombination and the search for homology. Recent studies have indicated that chromosomes increase their mobility after DNA damage. We have found that important genes and proteins like the RecA homolog and recombinase, RAD51, as well as the MRX complex contribute to this effect. By utilizing classic genetic approaches, we will further characterize the contributions to mobility and homology search of these and other factors. We will also examine the behavior of DNA ends during double-strand break formation to resolve long-standing questions about how these ends behave during repair. The combined experiments of this study will add significantly to our understanding of how DNA lesions are repaired.